Axon growth inhibitory CNS matrix proteins, such as tenascin-R (TN-R), have been supposed to contribute to the poor regenerative capacity of adult mammalian CNS. With regard to TN-R function in low vertebrates capable of CNS regeneration, questions of particular interest concern the (co)evolution of ligand-receptor pairs and cellular response mechanisms associated with axon growth inhibition and oligodendrocyte differentiation. We address here these questions in a series of comparative in vivo and in vitro analyses using TN-R proteins purified from different vertebrates (from fish to human). Our studies provide strong evidence that unlike TN-R of higher vertebrates, fish TN-R proteins are not repellent for fish and less repellent for mammalian neurons and do not interfere with F3/contactin- and fibronectin-mediated mammalian cell adhesion and axon growth. However, axonal repulsion is induced in fish neurons by mammalian TN-R proteins, suggesting that the intracellular inhibitory machinery induced by TN-R-F3 interactions is already present during early vertebrate evolution. In contrast to TN-R-F3, TN-R-sulfatide interactions, mediating oligodendrocyte adhesion and differentiation, are highly conserved during vertebrate evolution. Our findings thus indicate the necessity of being cautious about extrapolations of the function of ligand-receptor pairs beyond a species border and, therefore, about the phylogenetic conservation of a molecular function at the cellular/tissue level.

In this study, the neural phenotype is explored in rodent models of the spinocerebellar disorder known as the Friedreich Ataxia (FA), which results from mutations within the gene encoding the Frataxin mitochondrial protein. For this, the M12 line, bearing a targeted mutation, which disrupts the Frataxin gene exon 4 was used, together with the M02 line, which, in addition, is hemizygous for the human Frataxin gene mutation (Pook transgene), implying the occurrence of 82-190 GAA repeats within its first intron. The mutant mice phenotype was compared to the one of wild type littermates in regions undergoing differential profiles of neurogenesis, including the cerebellar cortex and the spinal cord by using neuronal (β-tubulin) and glial (Glial Fibrillary Acidic Protein) markers as well as the Contactin 1 axonal glycoprotein, involved in neurite growth control. Morphological/morphometric analyses revealed that while in Frataxin mutant mice the neuronal phenotype was significantly counteracted, a glial upregulation occurred at the same time. Furthermore, Contactin 1 downregulation suggested that changes in the underlying gene contributed to the disorder pathogenesis. Therefore, the FA phenotype implies an alteration of the developmental profile of neuronal and glial precursors. Finally, epigallocatechin gallate polyphenol administration counteracted the disorder, indicating protective effects of antioxidant administration.

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

Cell fate commitment of spinal progenitor neurons is initiated by long-range, midline-derived, morphogens that regulate an array of transcription factors that, in turn, act sequentially or in parallel to control neuronal differentiation. Included among these are transcription factors that regulate the expression of receptors for guidance cues, thereby determining axonal trajectories. The Ig/FNIII superfamily molecules TAG1/Axonin1/CNTN2 (TAG1) and Neurofascin (Nfasc) are co-expressed in numerous neuronal cell types in the CNS and PNS - for example motor, DRG and interneurons - both promote neurite outgrowth and both are required for the architecture and function of nodes of Ranvier. The genes encoding TAG1 and Nfasc are adjacent in the genome, an arrangement which is evolutionarily conserved. To study the transcriptional network that governs TAG1 and Nfasc expression in spinal motor and commissural neurons, we set out to identify cis elements that regulate their expression. Two evolutionarily conserved DNA modules, one located between the Nfasc and TAG1 genes and the second directly 5' to the first exon and encompassing the first intron of TAG1, were identified that direct complementary expression to the CNS and PNS, respectively, of the embryonic hindbrain and spinal cord. Sequential deletions and point mutations of the CNS enhancer element revealed a 130bp element containing three conserved E-boxes required for motor neuron expression. In combination, these two elements appear to recapitulate a major part of the pattern of TAG1 expression in the embryonic nervous system.

The Cntn1 (Contactin/F3/F11) cell adhesion molecule is involved in axon growth and guidance, fasciculation, synapse formation, and myelination in birds and mammals. We identified Cntn1 genes in goldfish, zebrafish, and fugu, and provide evidence for a fish-specific duplication leading to Cntn1a and Cntn1b. Our analyses suggest a subfunctionalization for the Cntn1 paralogs in zebrafish compared to other vertebrates which have a single Cntn1 gene. Similar to Cntn1a, Cntn1b transcripts are found in subsets of sensory and motor neurons. However, Cntn1b is detected later and more restricted than Cntn1a. This spatio-temporal expression pattern of the two zebrafish Cntn1 paralogs suggests functions related to those of mammalian Cntn1. In adult goldfish, Cntn1b is expressed in oligodendrocytes and is upregulated in retinal ganglion cells after optic nerve transection, which is consistent with an additional role during regeneration.

In this study, we address the activation profile of the gene encoding the mouse axonal glycoprotein F3/Contactin. Promoter sequences previously characterized in vitro are used to drive an Enhanced Green Fluorescent Protein reporter in transgenic mice. In developing cerebellum, differential transgene expression occurs within distinct cell populations. At P0 the transgene is activated in postmitotic granule neurons undergoing radial migration, a sharp upregulation occurring at P6-P8, with a gradual decline from this stage onward. In Purkinje cells, promoter activation, first detected at P3, peaks at around P6 and is fully downregulated by P16. The transgene is also expressed in Ng2- and O4-positive cells, mostly at the end of the first postnatal week, suggesting correlation with early oligodendrocyte differentiation. These data indicate that the complex organization of the regulatory region of the F3/Contactin gene is necessary for directing its articulated expression in different neural cells types and for its developmental function.

The expression of the cell recognition molecule F3/Contactin (CNTN1) is generally associated with the functions of post-mitotic neurons. In the embryonic cortex, however, we find it expressed by proliferating ventricular zone (VZ) precursors. In contrast to previous findings in the developing cerebellum, F3/Contactin transgenic overexpression in the early cortical VZ promotes proliferation and expands the precursor pool at the expense of neurogenesis. At later stages, when F3/Contactin levels subside, however, neurogenesis resumes, suggesting that F3/Contactin expression in the VZ is inversely related to neurogenesis and plays a role in a feedback control mechanism, regulating the orderly progression of cortical development. The modified F3/Contactin profile therefore results in delayed corticogenesis, as judged by downregulation in upper and lower layer marker expression and by BrdU birth dating, indicating that, in this transgenic model, increased F3/Contactin levels counteract neuronal precursor commitment. These effects also occur in primary cultures and are reproduced by addition of an F3/Fc fusion protein to wild type cultures. Together, these data indicate a completely novel function for F3/Contactin. Parallel changes in the generation of the Notch Intracellular Domain and in the expression of the Hes-1 transcription factor indicate that activation of the Notch pathway plays a role in this phenotype, consistent with previous in vitro reports that F3/Contactin is a Notch1 ligand.

In this study the mechanisms are explored, which modulate expression and function of cell surface adhesive glycoproteins of the Immunoglobulin Supergene Family (IgSF), and in particular of its Contactin subset, during neuronal precursor developmental events. In this context, a specific topic concerns the significance of the expression profile of such molecules and their ability to modulate signaling pathways activated through nutraceuticals, in particular polyphenols, administration. Both in vitro and in vivo approaches are chosen. As for the former, by using as a model the human SH-SY5Y neuroblastoma line, the effects of grape seed polyphenols are evaluated on proliferation and commitment/differentiation events along the neuronal lineage. In SH-SY5Y cell cultures, polyphenols were found to counteract precursor proliferation while promoting their differentiation, as deduced by studying their developmental parameters through the expression of cell cycle and neuronal commitment/differentiation markers as well as by measuring neurite growth. In such cultures, Cyclin E expression and BrdU incorporation were downregulated, indicating reduced precursor proliferation while increased neuronal differentiation was inferred from upregulation of cell cycle exit (p27-Kip) and neuronal commitment (NeuN) markers as well as by measuring neurite length through morphometric analysis. The polyphenol effects on developmental parameters were also explored in vivo, in cerebellar cortex, by using as a model the TAG/F3 transgenic line, which undergoes delayed neural development as a consequence of Contactin1 adhesive glycoprotein upregulation and premature expression under control of the Contactin2 gene (Cntn-2) promoter. In this transgenic line, a Notch pathway activation is known to occur and polyphenol treatment was found to counteract such an effect, demonstrated through downregulation of the Hes-1 transcription factor. Polyphenols also downregulated the expression of adhesive glycoproteins of the Contactin family themselves, demonstrated for both Contactin1 and Contactin2, indicating the involvement of changes in the expression of the underlying genes in the observed phenotype. These data support the hypothesis that the complex control exerted by polyphenols on neural development involves modulation of expression and function of the genes encoding cell adhesion molecules of the Contactin family and of the associated signaling pathways, indicating potential mechanisms whereby such compounds may control neurogenesis.

The amyotrophic lateral sclerosis (ALS) research community was one of the first to adopt methodology guidelines to improve preclinical research reproducibility. We here present the results of a systematic review to investigate how the standards in this field changed over the 10-year period during which the guidelines were first published (2007) and updated (2010).